Biologically Active Oxidized Phospholipids in Atherosclerosis

Article By Stanley L. Hazen, M.D., Ph.D., Kirk Maxey, M.D., & Eugene Podrez, M.D., Ph.D.

Atherosclerosis is responsible for one third of all deaths in North America, and for 80% of all deaths among diabetic patients.¹ The health risks of atherosclerosis will become even more dominant in the future, as obesity and adult onset diabetes become more prevalent. Atherosclerosis literally means the turning of the blood vessels to stone, easily seen in the calcified necrotic lesions of chronic disease. This attention to the physical manifestations of the disease continued into the recent past, as physicians and the public alike became fixated by the cholesterol contained within placque macrophages. More recently, scientists have recognized that foam cells and calcifications are both late manifestations of a chronic inflammatory disease.² The molecular details of early atherosclerotic inflammation begin with the activation of vascular endothelial cells, the increased transport of lipoprotein particles into the subendothelial extracellular space, and the oxidative modification of those particles by cellular mechanisms. This review will focus on the numerous, newly described lipid species that result from these initial steps, orchestrating the onset of atherosclerosis.

Inflammation and oxidative stress are key factors in atherogenesis contributing significantly to the initiation, progression, and rupture of lipid-rich atherosclerotic plaques. Associated with the inflammatory response and oxidative stress are lipid peroxidation and formation of bioactive lipids. Many bioactive lipids have been extracted and identified from plaque biopsy specimens and from plasma. These include oxidized phospholipids (oxPL), short chain reactive aldehydes, the potent inflammatory mediator platelet-activating factor (PAF), PAF analogs, oxidized cholesteryl esters, oxidized free fatty acids, lysophosphatidyl choline, oxysterols, and isoprostanes (see figures 1 and 2). Bioactive lipid peroxidation products derived from LDL and cell membranes are produced by activated cells. Release of esterified bioactive oxidation products is often facilitated via cleavage by phospholipases, or as a consequence of oxidative cleavage. Specifically, oxidized phospholipids are generated when LDL or cellular phospholipids containing polyunsaturated fatty acids (PUFAs) at the sn-2 position undergo oxidative attack. A common consequence of such attack is oxidative fragmentation of the sn-2 residue with generation of phospholipid molecular species containing shortened sn-2 residues. Other products generated during the peroxidation of PUFA are free short chain reactive aldehydes, such as dodecadienal (DDA). These aldehydes may rapidly react with proteins forming covalent adducts. Accumulation of oxidatively modified proteins in cellular targets and tissues occurs during aging, oxidative stress, and in a variety of diseases including atherosclerosis. 4-Hydroxy-2-alkenals are prominent aldehyde substances generated during the peroxidation of PUFA. 4-hydroxy-2-Nonenal (4-HNE) is a major aldehyde formed during the lipid peroxidation of ω-6 PUFAs such as linoleic acid and arachidonic acid. On the other hand, the peroxidation of ω-3 PUFAs, such as docosahexaenoic acid and eicosapentaenoic acid, generates a closely related compound, 4-hydroxy-2-hexenal (HHE). Specific antibodies against protein-bound 4-HNE or HHE have been found in atherosclerotic lesions.

Biologically active oxPL initiate and modulate many of the cellular events seen in the developing fatty streak. Complex mixtures of oxPL, or specific synthetic oxPL induce an inflammatory response in cells by the induction of proinflammatory genes (such as MCP-1, IL-8, tissue factor, and others). In human aortic endothelial cells, oxPL change the expression of a number of genes related toangiogenesis, atherosclerosis, inflammation, and wound healing. Inaddition oxPL activate platelets, induce differentiation of monocytes andinduce dedifferentiation of smooth muscle cells - processes related toplaque formation. oxPL act via transcription factors such as peroxisomeproliferator-activated receptors (PPARs) α and γ, and via nuclear factor of activated T cells (NFAT), and Egr-1. They also modulate the fate of aninflammatory response by intervening into such processes as removal ofapoptotic cells and by dampening bacterial-induced inflammation. Finally, recent studies have shown that a select group of oxPL serve as ligands forscavenger receptors, thus mediating highly specific cellular recognition and internalization of targets containing complex mixtures ofbiologically active oxidized lipids. Thus, a growing body of evidence suggests that oxPL may play a general role in inflammation and related processes such as atherosclerosis.

There is considerable in vivo evidence that oxPL play a significant rolein the development of atherosclerosis. oxPL accumulation in vivo correlateswith the development of atherosclerosis. Bioactive oxPL are elevated inthe circulation in two major mouse models of atherosclerosis: ApoE^(-/-) and LDLr^(-/-) mice on the Western diet.³ Pharmacological inhibition ofbioactive oxPL in vivo by WEB 2086 diminishes formation of fatty streaksin fat-fed mice.⁴ oxPL accumulate in HDL and this accumulation is associated with changes in HDL capability to promote cholesterol effluxfrom cells.5 Studies of two HDL associated enzymes, serum paraoxonase (PON1) and PAF-acetylhydrolase (PAF-AH), that are responsible for hydrolysis of plasma oxPL^3,6 indirectly support the role of oxPL in atherosclerosis. Both enzymes play an a theroprotective role in vivo. Mice deficient for paraoxonase accumulate oxPL and develop more lesions than control mice.^7,8 These data demonstrate an association between PON1 deficiency, lipoprotein phospholipid oxidation, and atherogenesis in apo Eknockout mice. In humans, polymorphisms in the PON1 gene and in PAFAH gene have been associated with the risk for coronary artery disease.^9,10 PAF-AH gene transfer in apolipoprotein E-deficient mice reduced oxidized lipoproteins, reduced lipoprotein-induced macrophage adhesion, and reduced macrophage homing and smooth muscle cell accumulation in the arterial wall. This resulted in a significant reduction of the neointimal area and atherosclerotic lesions.^11,12 Taken together these results suggest that oxPL contribute significantly to atherogenesis. Oxidized phospholipids that are believed to play a role in atherogenesis, including several recently identified species, and their biological activities are described below.

Endothelial Cell Activators

Two specific oxPL, 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (POV-PC) and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphorylcholine (PGPC), were identified as abundant products in LDL oxidized by various methods and were shown to have a major role in the activation of endothelial cells and induction of leukocyte binding. POV-PC and PGPC are found in the circulation and in atherosclerotic lesions. POV-PC is a potent regulator of monocyte-specific endothelial interactions. It increases monocyte binding by inducing the surface expression of the connecting segment 1 domain of fibronectin, but does not increase neutrophil binding. In addition, POV-PC strongly inhibits lipopolysaccharide-mediated induction of neutrophil binding and expression of E-selectin protein and mRNA. This inhibition is mediated by a protein kinase A-dependent pathway. It is also suggested that POV-PC stimulates a cAMP-mediated pathway. In contrast to POV-PC, PGPC induces both monocyte and neutrophil binding and expression of E-selectin and vascular cell adhesion molecule-1. It is suggested that these two phospholipids act by different unidentified novel receptors. It was also shown that the group at the sn-2 position determines the specific bioactivity of POV-PC/PGPC and that the substitution of stearoyl for palmitoyl at the sn-1 position or ethanolamine for choline at the sn-3 position of the phospholipid did not alter bioactivity.

Oxidized Phospholipids That are Ligands for the Macrophage Scavenger Receptor CD36

The early cellular hallmark of the atherosclerotic process is an uptake and accumulation of LDL cholesterol by arterial macrophages, leading to foam cell formation. Cells like macrophages are normally protected from the toxic effects of excess cholesterol by multiple mechanisms, including the down-regulation of surface LDL receptor molecules in response to replete intracellular cholesterol stores. In contrast, LDL which is first oxidized or chemically modified may be taken up by scavenger receptors, whose surface expression is not diminished upon exposure to excess cholesterol. A variety of scavenger receptors expressed on macrophages have been described. Among these, the scavenger receptor class A (SR-A) and CD36 were demonstrated to be major receptors responsible for the uptake of modified forms of LDL in vitro and are involved in vivo in a mouse model of atherosclerosis. It was established previously that SR-A recognizes the protein moiety of modified LDL. The exact molecular structure(s) of the ligand(s) recognized by CD36 were not known until recently. A role for CD36 as the receptor responsible for the recognition of oxidized lipids extracted from Cu²⁺-oxLDL was shown.^13,14 oxPL covalently linked to apolipoprotein B-100 in extensively-oxidized LDL (e.g., Cu²⁺-oxLDL) have also been suggested to serve as ligands for CD36.^13,15,16 It was later found that POV-PC which reacted with a peptide yielding a Schiff base, or POV-PC that underwent an aldol type self-condensation, represents an immunogen for an antibody (EO6) that blocks the binding of copper-oxidized LDL to CD36.¹⁷ Based on this information it was concluded that such a product represents a ligand for CD36.

A combination of mass spectrometry and both analytical and synthetic chemistry was employed recently to isolate and structurally define a novel family of oxidized choline glycerophospholipids (oxPC_CD36) that, in an unbound state, serve as specific high affinity ligands for CD36.¹⁸ Four major structurally-related phospholipids with CD36 binding activity (oxPC_CD36) were identified from oxidized 1-hexadecanoyl-2-eicosatetra-5’,8’,11’,14’-enoyl-sn-glycero-3-phosphocholine (PAPC), and four corresponding structural analogs with CD36 binding activity were identified from oxidized 1-hexadecanoyl-2-octadecadi-9’,12’-enoyl-sn-glycero-3-phosphocholine (PLPC). LC/ESI/MS/MS studies demonstrated their formation during the oxidization of LDL by multiple distinct pathways and their participation in CD36 mediated recognition of different forms of oxidized LDL.18 The critical structural elements required for oxidized phospholipids to serve as ligands for CD36 were defined, namely a phospholipid with a truncated sn-2 acyl group that incorporates a terminal hydroxy (or oxo) unsaturated carbonyl.¹⁸ A representative example is KOdiA-PC (Fig. 1). CD36 is the major receptor on macrophages mediating the recognition of oxPC_CD36 species. oxPC_CD36 promote CD36-dependent recognition when present at only a few molecules per particle, resulting in macrophage binding, uptake, metabolism, and foam cell formation. oxPC_CD36 can transfer from oxidized to unoxidized LDL or other lipid containing targets and thus induce CD36 recognition.19 oxPC_CD36 are generated in vivo and are enriched in atherosclerotic lesions. These results suggested that formation of this novel family of oxidized phospholipids might play an important role in CD36-mediated recognition of oxidized lipoproteins, senescent or apoptotic cells, and foam cell formation.

PAF and PAF Mimetics

PAF is a biologically active phospholipid, structurally identified as 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine. PAF is synthesized from a specific subclass of phosphatidylcholines that contains an ether, rather than an ester, bond at the sn-1 position of the glycerol backbone. This phospholipid precursor is a minor component of low-density LDL. Typically, a PUFA such as arachidonate is found at the 2-position in this subclass of phosphatidylcholine. Hydrolysis of this precursor phospholipid and subsequent acetylation of the reaction product, alkyl lyso-phosphatidylcholine, with acetyl-CoA generates PAF. Both reactions in this PAF synthetic path are tightly regulated. Thus, the physiologic synthesis of PAF is closely controlled. PAF induces its inflammatory response through a single G-protein coupled receptor,²⁰ and does so at subnanomolar concentrations. The PAF receptor shows several hundred-fold selectivity for the sn-1 ether bond of PAF, and complete specificity for the sn-2 acetyl residue compared with the long chain fatty acyl residue of most alkyl phosphatidylcholines. The choline headgroup confers a several thousand-fold advantage over the related phosphatidylethanolamine analog. Some of the inflammatory actions of PAF include platelet aggregation, hypotension, anaphylactic shock, and increased vascular permeability.²¹ PAF also induces atherogenic effects by activating monocytes and smooth muscle cell growth.^22,23

In contrast to the tightly regulated physiologic generation of PAF, uncontrolled processes of free radical oxidation generate PAF analogs in vivo and in vitro. Since precursors for PAF synthesis frequently contain arachidonate in the sn-2 position, they can also be a target for peroxidation. One outcome of this type of uncontrolled chemical reaction is the fragmentation of the residue at the sn-2 position. As a result of this fragmentation, some of the cellular or LDL phospholipid oxidation products structurally resemble PAF. These oxidatively-generated PAF mimetics stimulate monocytes,²⁴ leukocytes,²⁵ and platelets.²⁶ They are found in atherosclerotic lesions or even in blood after exposure to cigarette smoke.^23,27 All of the PAF receptor agonists generated during the oxidation of LDL are derived from the oxidation of the alkyl phosphatidylcholines found in LDL. Major bioactive species generated by oxidative fragmentation of alkyl phosphatidylcholines are butanoyl- and butenoyl-PAF. Thus, oxidation of rare phospholipid species in LDL generates bioactive, short chain PAF-analogs.

Azelaoyl PAF - A Hybrid Oxidized Phospholipid

An abundant oxidatively fragmented phospholipid in oxLDL was recently described that possessed an ether bond in the sn-1 position and a short-chain diacid group in the sn-2 position (hexadecyl azelaoyl phosphatidylcholine or Azelaoyl PAF (AzPAF).²⁸ AzPAF serves as a specific high affinity ligand and agonist for PPARγ and is recognized by CD36. It binds to the ligand binding pocket of recombinant PPARγ with an affinity that is equivalent to rosiglitazone. AzPAF induces PPRE reporter gene expression, with a half-maximal effect at 100 nM. It induces expression of CD36 and cyclooxygenase-2 in primary human monocytes via the PPAR-responsive element present in the corresponding genes. PPARγ activity of AzPAF depends on an ether bond since the diacyl homolog of AzPAF, 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PAz-PC), is about 100-fold less effective than AzPAF. CD36-binding activity of AzPAF probably depends on the sn-2 residue, since according to our studies an ether bond is not required for CD36 binding activity¹⁸ suggesting that PAz-PC is a ligand for CD36. Circulating human monocytes do not contain PPARγ, but PPARγ is induced rapidly in primary monocytes by appropriate outside-in signaling, sensitizing them to previously undetectable agonists in oxidized LDL. Increased expression of CD36 in monocytes in turn aids the accumulation of AzPAF in cells. Thus, CD36 helps AzPAF to carry out its PPARγ activating ability,²⁸ while AzPAF supports the expression of CD36. These findings establish a new link connecting fragmented phospholipids of oxidized LDL with the induction of PPAR-regulated genes, in monocytes.

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